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Arterial stiffening occurs during natural aging, is associated with an increased risk of adverse cardiovascular events, and can follow different timelines in males and females. One mechanism of arterial stiffening includes remodeling of the extracellular matrix (ECM), which alters the wall material properties. We used elastin haploinsufficient (Eln+/-) and wildtype (Eln+/+) mice to investigate how material properties of two different arteries (ascending aorta and carotid artery) change with age, sex, and ECM composition. We used a constitutive model by Dong and Sun that is based on the Holzapfel-Gasser-Ogden (HGO) type, but does not require a discrete number of fibrous ECM families and allows varied deformation coupling. We find that the amount of deformation coupling for the best fit model depends on the artery type. We also find that remodeling to maintain homeostatic (i.e., young, wildtype) values of biomechanical parameters with age, sex, and ECM composition depends on the artery type, with ascending aorta being more adaptable than carotid artery. Fitted material constants indicate sex-dependent remodeling that may be important for determining the time course of arterial stiffening in males and females. We correlated fitted material constants with ECM composition measured by biochemical (ascending aorta) or histological (carotid artery) methods. We show significant correlations between ECM composition and material parameters for the mean values for each group, with biochemical measurements correlating more strongly than histological measurements. Understanding how arterial stiffening depends on age, sex, ECM composition, and artery type may help design effective, personalized clinical treatment strategies., (Copyright © 2024 by ASME.)

Aortic wall material properties are needed for computational models and for comparisons across developmental and disease states. There has been abundant work in comparing aortic material properties across disease states, but limited work across developmental states. We performed passive biaxial mechanical testing on newborn mouse aorta with (Eln +/+ ) and without (Eln -/- ) elastin. Elastin provides elasticity to the aortic wall and is necessary for survival beyond birth in the mouse. Mechanically functional elastin is challenging to create in vitro and so Eln -/- aorta can be a comparison for tissue engineered arteries with limited elastin amounts. We found that a traditional arterial strain energy function provided reasonable fits to newborn mouse aorta and generally predicted lower material constants in Eln -/- compared to Eln +/+ aorta. At physiologic pressures, the circumferential stresses and moduli trended lower in Eln -/- compared to Eln +/+ aorta. Increased blood pressure in Eln -/- mice helps to alleviate the differences in stresses and moduli. Increased blood pressure also serves to partially offload stresses in the isotropic compared to the anisotropic component of the wall. The baseline material parameters can be used in computational models of growth and remodeling to improve understanding of developmental mechanobiology and tissue engineering strategies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models., Conclusions: These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Elastin (ELN) insufficiency leads to the cardiovascular hallmarks of the contiguous gene deletion disorder, Williams-Beuren syndrome, including hypertension and vascular stiffness. Previous studies showed that Williams-Beuren syndrome deletions, which extended to include the NCF1 gene, were associated with lower blood pressure (BP) and reduced vascular stiffness. NCF1 encodes for p47phox, the regulatory component of the NOX1 NADPH oxidase complex that generates reactive oxygen species (ROS) in the vascular wall. Dihydroethidium and 8-hydroxyguanosine staining of mouse aortas confirmed that Eln heterozygotes ( Eln +/- ) had greater ROS levels than the wild-types ( Eln +/+ ), a finding that was negated in vessels cultured without hemodynamic stressors. To analyze the Nox effect on ELN insufficiency, we used both genetic and chemical manipulations. Both Ncf1 haploinsufficiency ( Ncf1 +/- ) and Nox1 insufficiency ( Nox1 -/y ) decreased oxidative stress and systolic BP in Eln +/- without modifying vascular structure. Chronic treatment with apocynin, a p47phox inhibitor, lowered systolic BP in Eln +/- , but had no impact on Eln +/+ controls. In vivo dosing with phenylephrine (PE) produced an augmented BP response in Eln +/- relative to Eln +/+ , and genetic modifications or drug-based interventions that lower Nox1 expression reduced the hypercontractile response to PE in Eln +/- mice to Eln +/+ levels. These results indicate that the mechanical and structural differences caused by ELN insufficiency leading to oscillatory flow can perpetuate oxidative stress conditions, which are linked to hypertension, and that by lowering the Nox1-mediated capacity for vascular ROS production, BP differences can be normalized., (Published by Oxford University Press on behalf of American Physiological Society 2021. This work is written by US Government employees and is in the public domain in the US.)

Elastin is a primary structural protein in the arterial wall that contributes to vascular mechanical properties and degrades with aging. Aging is associated with arterial stiffening and an increase in blood pressure. There is evidence that arterial aging follows different timelines with sex. Our objective was to investigate how elastin content affects arterial remodeling in male and female mice with aging. We used male and female wild-type ( Eln +/+ ) and elastin heterozygous ( Eln +/- ) mice at 6, 12, and 24 mo of age and measured their blood pressure and arterial morphology, wall structure, protein content, circumferential stress, stretch ratio, and stiffness. Two arteries were used with varying contents of elastin: the left common carotid and ascending aorta. We show that Eln +/- arteries start at a different homeostatic set point for circumferential wall stress, stretch, and material stiffness but show similar increases with aging to Eln +/+ mice. With aging, structural stiffness is greatly increased, while material stiffness and circumferential stress are only slightly increased, highlighting the importance of maintaining these homeostatic values. Circumferential stretch shows the smallest change with age and may be important for controlling cellular phenotype. Independent sex differences are mostly associated with males being larger than females; however, many of the measured factors show age × sex and/or genotype × sex interactions, indicating that males and females follow different cardiovascular remodeling timelines with aging and are differentially affected by reduced elastin content. NEW & NOTEWORTHY A comprehensive study on arterial mechanical behavior as a function of elastin content, aging, and sex in mice. Elastin haploinsufficient arteries start at a different homeostatic set point for mechanical parameters such as circumferential stress, stretch, and material stiffness. Structural stiffness of the arterial wall greatly increases with aging, as expected, but there are interactions between sex and aging for most of the mechanical parameters that are important to consider in future work.

Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 ( Fbln4 -/- ) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4 -/- cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Supravalvular aortic stenosis (SVAS) is a narrowing of the aorta caused by elastin (ELN) haploinsufficiency. SVAS severity varies among patients with Williams-Beuren syndrome (WBS), a rare disorder that removes one copy of ELN and 25-27 other genes. Twenty percent of children with WBS require one or more invasive and often risky procedures to correct the defect while 30% have no appreciable stenosis, despite sharing the same basic genetic lesion. There is no known medical therapy. Consequently, identifying genes that modify SVAS offers the potential for novel modifier-based therapeutics. To improve statistical power in our rare-disease cohort (N = 104 exomes), we utilized extreme-phenotype cohorting, functional variant filtration and pathway-based analysis. Gene set enrichment analysis of exome-wide association data identified increased adaptive immune system variant burden among genes associated with SVAS severity. Additional enrichment, using only potentially pathogenic variants known to differ in frequency between the extreme phenotype subsets, identified significant association of SVAS severity with not only immune pathway genes, but also genes involved with the extracellular matrix, G protein-coupled receptor signaling and lipid metabolism using both SKAT-O and RQTest. Complementary studies in Eln+/-; Rag1-/- mice, which lack a functional adaptive immune system, showed improvement in cardiovascular features of ELN insufficiency. Similarly, studies in mixed background Eln+/- mice confirmed that variations in genes that increase elastic fiber deposition also had positive impact on aortic caliber. By using tools to improve statistical power in combination with orthogonal analyses in mice, we detected four main pathways that contribute to SVAS risk., (Published by Oxford University Press 2020.)

Deposition of elastin and collagen in the aorta correlates with increases in blood pressure and flow during development, suggesting that the aorta adjusts its mechanical properties in response to hemodynamic stresses. Elastin knockout (Eln -/- ) mice have high blood pressure and pathological remodeling of the aorta and die soon after birth. We hypothesized that decreasing blood pressure in Eln -/- mice during development may reduce hemodynamic stresses and alleviate pathological remodeling of the aorta. We treated Eln +/+ and Eln -/- mice with the anti-hypertensive medication captopril throughout embryonic development and then evaluated left ventricular (LV) pressure and aortic remodeling at birth. We found that captopril treatment decreased Eln -/- LV pressure to values near Eln +/+ mice and alleviated the wall thickening and changes in mechanical behavior observed in untreated Eln -/- aorta. The changes in thickness and mechanical behavior in captopril-treated Eln -/- aorta were not due to alterations in measured elastin or collagen amounts, but may have been caused by alterations in smooth muscle cell (SMC) properties. We used a constitutive model to understand how changes in stress contributions of each wall component could explain the observed changes in composite mechanical behavior. Our modeling results show that alterations in the collagen natural configuration and SMC properties in the absence of elastin may explain untreated Eln -/- aortic behavior and that partial rescue of the SMC properties may account for captopril-treated Eln -/- aortic behavior.

Rationale: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown., Objective: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries., Methods and Results: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries., Conclusions: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.

Studies over the years have described a filamentous structure to mature elastin that suggests a complicated packing arrangement of tropoelastin subunits. The currently accepted mechanism for tropoelastin assembly requires microfibrils to serve as a physical extracellular scaffold for alignment of tropoelastin monomers during and before crosslinking. However, recent evidence suggests that the initial stages of tropoelastin assembly occur within the cell or at unique assembly sites on the plasma membrane where tropoelastin self assembles to form elastin aggregates. Outside the cell, elastin aggregates transfer to growing elastic fibers in the extracellular matrix where tensional forces on microfibrils generated through cell movement help shape the growing fiber. Overall, these observations challenge the widely held idea that interaction between monomeric tropoelastin and microfibrils is a requirement for elastin assembly, and point to self-assembly of tropoelastin as a driving force in elastin maturation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Elastin is expressed in most tissues that require elastic recoil. The protein first appeared coincident with the closed circulatory system, and was critical for the evolutionary success of the vertebrate lineage. Elastin is expressed by multiple cell types in the lung, including mesothelial cells in the pleura, smooth muscle cells in airways and blood vessels, endothelial cells, and interstitial fibroblasts. This highly crosslinked protein associates with fibrillin-containing microfibrils to form the elastic fiber, which is the physiological structure that functions in the extracellular matrix. Elastic fibers can be woven into many different shapes depending on the mechanical needs of the tissue. In large pulmonary vessels, for example, elastin forms continuous sheets, or lamellae, that separate smooth muscle layers. Outside of the vasculature, elastic fibers form an extensive fiber network that originates in the central bronchi and inserts into the distal airspaces and visceral pleura. The fibrous cables form a looping system that encircle the alveolar ducts and terminal air spaces and ensures that applied force is transmitted equally to all parts of the lung. Normal lung function depends on proper secretion and assembly of elastin, and either inhibition of elastin fiber assembly or degradation of existing elastin results in lung dysfunction and disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lung alveolarization requires precise coordination of cell growth with extracellular matrix (ECM) synthesis and deposition. The role of extracellular matrices in alveogenesis is not fully understood, because prior knowledge is largely extrapolated from two-dimensional structural analysis. Herein, we studied temporospatial changes of two important ECM proteins, laminin and elastin that are tightly associated with alveolar capillary growth and lung elastic recoil respectively, during both mouse and human lung alveolarization. By combining protein immunofluorescence staining with two- and three-dimensional imaging, we found that the laminin network was simplified along with the thinning of septal walls during alveogenesis, and more tightly associated with alveolar endothelial cells in matured lung. In contrast, elastin fibers were initially localized to the saccular openings of nascent alveoli, forming a ring-like structure. Then, throughout alveolar growth, the number of such alveolar mouth ring-like structures increased, while the relative ring size decreased. These rings were interconnected via additional elastin fibers. The apparent patches and dots of elastin at the tips of alveolar septae found in two-dimensional images were cross sections of elastin ring fibers in the three-dimension. Thus, the previous concept that deposition of elastin at alveolar tips drives septal inward growth may potentially be conceptually challenged by our data.

The functional form of elastin is a highly cross-linked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of the mature protein is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove nonelastin "contaminates" are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis require careful attention during purification and in tropoelastin-based assays. This chapter describes the most common approaches for purification of elastin and for preparing solubilized forms of the protein., (© 2018 Elsevier Inc. All rights reserved.)

Elastin and collagen levels in tissues are frequently difficult to measure because of each protein's limited solubility. This chapter provides detailed methodology for the determination of elastin, collagen, and total protein levels in a single tissue sample. All three assays start with an acid hydrolysate of the tissue, which breaks the tissue-associated proteins down to their component amino acids. Marker amino acids unique to each protein (desmosine for elastin and hydroxyproline for collagen) are then quantified. Total protein content, useful as a denominator for data normalization, can also be measured from a portion of the hydrolysate using an assay for free amino groups. These measurements are performed using convenient 96-well assay plates and require only a plate reader to determine absorbance., (© 2018 Elsevier Inc. All rights reserved.)

Elastic fibers are present in low quantities in tendon, where they are located both within fascicles near tenocytes and more broadly in the interfascicular matrix (IFM). While elastic fibers have long been known to be significant in the mechanics of elastin-rich tissue (i.e., vasculature, skin, lungs), recent studies have suggested a mechanical role for elastic fibers in tendons that is dependent on specific tendon function. However, the exact contribution of elastin to properties of different types of tendons (e.g., positional, energy-storing) remains unknown. Therefore, this study purposed to evaluate the role of elastin in the mechanical properties and collagen alignment of functionally distinct supraspinatus tendons (SSTs) and Achilles tendons (ATs) from elastin haploinsufficient (HET) and wild type (WT) mice. Despite the significant decrease in elastin in HET tendons, a slight increase in linear stiffness of both tendons was the only significant mechanical effect of elastin haploinsufficiency. Additionally, there were significant changes in collagen nanostructure and subtle alteration to collagen alignment in the AT but not the SST. Hence, elastin may play only a minor role in tendon mechanical properties. Alternatively, larger changes to tendon mechanics may have been mitigated by developmental compensation of HET tendons and/or the role of elastic fibers may be less prominent in smaller mouse tendons compared to the larger bovine and human tendons evaluated in previous studies. Further research will be necessary to fully elucidate the influence of various elastic fiber components on structure-function relationships in functionally distinct tendons.

Objective: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN , the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined., Approach and Results: We quantified determinants of luminal stenosis in thoracic aortas of Eln -/- . Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject.ELN . Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject., Conclusions: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency., (© 2017 American Heart Association, Inc.)

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd., (Copyright © 2014 John Wiley & Sons, Ltd.)

Elastin (Eln) insufficiency in mice and humans is associated with hypertension and altered structure and mechanical properties of large arteries. However, it is not known to what extent functional or structural changes in resistance arteries contribute to the elevated blood pressure that is characteristic of Eln insufficiency. Here, we investigated how Eln insufficiency affects the structure and function of the resistance vasculature. A functional profile of resistance vasculature in Eln(+/-) mice was generated by assessing small mesenteric artery (MA) contractile and vasodilatory responses to vasoactive agents. We found that Eln haploinsufficiency had a modest effect on phenylephrine-induced vasoconstriction, whereas ANG II-evoked vasoconstriction was markedly increased. Blockade of ANG II type 2 receptors with PD-123319 or modulation of Rho kinase activity with the inhibitor Y-27632 attenuated the augmented vasoconstriction, whereas acute Y-27632 administration normalized blood pressure in Eln(+/-) mice. Sodium nitroprusside- and isoproterenol-induced vasodilatation were normal, whereas ACh-induced vasodilatation was severely impaired in Eln(+/-) MAs. Histologically, the number of smooth muscle layers did not change in Eln(+/-) MAs; however, an additional discontinuous layer of Eln appeared between the smooth muscle layers that was absent in wild-type arteries. We conclude that high blood pressure arising from Eln insufficiency is due partly to permanent changes in vascular tone as a result of increased sensitivity of the resistance vasculature to circulating ANG II and to impaired vasodilatory mechanisms arising from endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation. Eln insufficiency causes augmented ANG II-induced vasoconstriction in part through a novel mechanism that facilitates contraction evoked by ANG II type 2 receptors and altered G protein signaling.

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Large artery stiffness, as measured by pulse wave velocity, is correlated with high blood pressure and may be a causative factor in essential hypertension. The extracellular matrix components, specifically the mix of elastin and collagen in the vessel wall, determine the passive mechanical properties of the large arteries. Elastin is organized into elastic fibers in the wall during arterial development in a complex process that requires spatial and temporal coordination of numerous proteins. The elastic fibers last the lifetime of the organism but are subject to proteolytic degradation and chemical alterations that change their mechanical properties. This review discusses how alterations in the amount, assembly, organization, or chemical properties of the elastic fibers affect arterial stiffness and blood pressure. Strategies for encouraging or reversing alterations to the elastic fibers are addressed. Methods for determining the efficacy of these strategies, by measuring elastin amounts and arterial stiffness, are summarized. Therapies that have a direct effect on arterial stiffness through alterations to the elastic fibers in the wall may be an effective treatment for essential hypertension.

Objective: The goal of this study was to determine whether antagonizing microRNA (miR)-29 enhances elastin (ELN) levels in cells and tissues lacking ELN., Methods and Results: miR-29 mimics reduced ELN levels in fibroblasts and smooth muscle cells, whereas miR-29 inhibition increased ELN levels. Antagonism of miR-29 also increased ELN levels in cells from patients haploinsufficient for ELN and in bioengineered human vessels., Conclusion: miR-29 antagonism may promote increased ELN levels during conditions of ELN deficiencies.

Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln(+/-) mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln(+/-), two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln(+/-)x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln(+/-) animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln(+/-)x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln(+/-), including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln(+/-) aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation.

Introduction: Elastin fibers confer passive recoil to many tissues including the lung, skin, and arteries. In the penis, elastin is present in sinusoids, arterioles, and in the tunica albuginea. Although decreased penile elastin has been reported in men with erectile dysfunction, the exact role of elastin in physiologic processes integral to erection remains speculative., Aim: The aim of this study was to characterize erectile function in elastin-deficient mice., Methods: Elastin haploinsufficient mice (Eln(+/-) ) and aged match Eln(+/+) (Wt) mice were used. Cavernosum was removed from some mice for quantification of elastin, collagen, and smooth muscle actin. Ex vivo assessment of contractile force generation was performed by myography. In vivo assessment of intracorporal pressure normalized to mean arterial pressure in response to electrical stimulation of the cavernosal nerve was measured. Veno-occlusive function was determined by cavernosography., Main Outcome Measures: The main outcome measures of this study were the in vitro and in vivo assessment of cavernosal vasoreactivity, veno-occlusive function and erection in mice deficient in elastin., Results: Eln (+/-) mice exhibited ∼33% less penile elastin than Wt mice, with no change in collagen. Cavernosal tissue from Eln(+/-) mice has a significantly heightened contractile response, explained in part by increased smooth muscle cell content. Veno-occlusive function was significantly altered in Eln(+/-) mice. Interestingly, erectile function was impaired only at submaximal voltage (1 V) stimulation (there was no impairment during the higher 2-V stimulus)., Conclusions: Eln (+/-) mice display a cavernosal phenotype consistent with developmental changes attributable to the loss of elastin. These alterations confer a degree of altered erectile function that is able to be overridden by maximal stimulatory input. Altogether, these data suggest that elastin is important for erectile function., (© 2011 International Society for Sexual Medicine.)

Increased arterial stiffness and blood pressure are characteristic of humans and adult mice with reduced elastin levels caused by aging or genetic disease. Direct associations have been shown between increased arterial stiffness and hypertension in humans, but it is not known whether changes in mechanical properties or increased blood pressure occur first. Using genetically modified mice with elastin haploinsufficiency (Eln(+/-)), we investigated the temporal relationship between arterial mechanical properties and blood pressure throughout postnatal development. Our results show that some mechanical properties are maintained constant regardless of elastin amounts. The peak diameter compliance for both genotypes occurs near the physiologic pressure at each age, which acts to provide maximum pulse dampening. The stress-strain relationships are similar between genotypes and become nonlinear near the systolic pressure for each age, which serves to limit distension under high pressure. Our results also show that some mechanical properties are affected by reduced elastin levels and that these changes occur before measurable changes in blood pressure. Eln(+/-) mice have decreased aortic diameter and compliance in ex vivo tests that are significant by postnatal day 7 and increased blood pressure that is not significant until postnatal day 14. This temporal relationship suggests that targeting large arteries to increase diameter or compliance may be an effective treatment for human hypertension.

Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28-34 of the ELN gene in five probands with ADCL features and found five de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5), and c.2124del25 (CL-6). Four probands (CL-1,-2,-3,-6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGF-β) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications, associated with increased TGF-β signaling and mutation-specific differences in endoplasmic reticulum stress and apoptosis., (© 2011 Wiley-Liss, Inc.)

Objectives: To investigate the effects of a heterozygous elastin gene (Eln) abnormality (deletion of one Eln allele) on the structural characteristics of the vocal fold lamina propria using a mouse model of human disease., Study Design: Cross-sectional between-subjects design., Methods: Five mice, four with heterozygous Eln deletions (Eln +/-) serving as an animal model for the human disease supravalvular aortic stenosis and one normal wild-type control (Eln +/+) were used for this study. Vocal folds were obtained from each animal and stained for the protein elastin using histochemical methods. Descriptive data from qualitative visual inspection and quantitative data from microscopic digital image analysis were collected to determine the staining density of elastic fibers within the vocal fold lamina propria., Results: Qualitative visual inspection revealed greater staining density (eg, a greater quantity) for elastic fibers in the Eln +/+ animal. Quantitative measurements using digital pixel analysis of staining density revealed significant differences between mice with the two genotypes, confirming the qualitative findings., Conclusions: Results suggest that Eln requires two functioning alleles for normal structural development of the vocal fold lamina propria. This pilot evidence supports the hypothesis of a structural etiology causing altered vocal function in humans with a similar genotype., (Copyright © 2011 The Voice Foundation. Published by Mosby, Inc. All rights reserved.)

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rationale: Elastin is a ubiquitous extracellular matrix protein that is highly organized in heart valves and arteries. Because elastic fiber abnormalities are a central feature of degenerative valve disease, we hypothesized that elastin-insufficient mice would manifest viable heart valve disease., Objective: To analyze valve structure and function in elastin-insufficient mice (Eln(+/-)) at neonatal, juvenile, adult, and aged adult stages., Methods and Results: At birth, histochemical analysis demonstrated normal extracellular matrix organization in contrast to the aorta. However, at juvenile and adult stages, thin elongated valves with extracellular matrix disorganization, including elastin fragment infiltration of the annulus, were observed. The valve phenotype worsened by the aged adult stage with overgrowth and proteoglycan replacement of the valve annulus. The progressive nature of elastin insufficiency was also shown by aortic mechanical testing that demonstrated incrementally abnormal tensile stiffness from juvenile to adult stages. Eln(+/-) mice demonstrated increased valve interstitial cell proliferation at the neonatal stage and varied valve interstitial cell activation at early and late stages. Gene expression profile analysis identified decreased transforming growth factor-beta-mediated fibrogenesis signaling in Eln(+/-) valve tissue. Juvenile Eln(+/-) mice demonstrated normal valve function, but progressive valve disease (predominantly aortic regurgitation) was identified in 17% of adult and 70% of aged adult Eln(+/-) mice by echocardiography., Conclusions: These results identify the Eln(+/-) mouse as a model of latent aortic valve disease and establish a role for elastin dysregulation in valve pathogenesis.

Elastin is an essential component of vertebrate arteries that provides elasticity and stores energy during the cardiac cycle. Elastin production in the arterial wall begins midgestation but increases rapidly during the last third of human and mouse development, just as blood pressure and cardiac output increase sharply. The aim of this study is to characterize the structure, hemodynamics, and mechanics of developing arteries with reduced elastin levels and determine the critical time period where elastin is required in the vertebrate cardiovascular system. Mice that lack elastin (Eln(-/-)) or have approximately one-half the normal level (Eln(+/-)) show relatively normal cardiovascular development up to embryonic day (E) 18 as assessed by arterial morphology, left ventricular blood pressure, and cardiac function. Previous work showed that just a few days later, at birth, Eln(-/-) mice die with high blood pressure and tortuous, stenotic arteries. During this period from E18 to birth, Eln(+/-) mice add extra layers of smooth muscle cells to the vessel wall and have a mean blood pressure 25% higher than wild-type animals. These findings demonstrate that elastin is only necessary for normal cardiovascular structure and function in mice starting in the last few days of fetal development. The large increases in blood pressure during this period may push hemodynamic forces over a critical threshold where elastin becomes required for cardiovascular function. Understanding the interplay between elastin amounts and hemodynamic forces in developing vessels will help design treatments for human elastinopathies and optimize protocols for tissue engineering.

Elastin is the polymeric protein responsible for the properties of extensibility and elastic recoil of the extracellular matrix in a variety of tissues. Although proper assembly of the elastic matrix is crucial for its durability, the process by which this assembly takes place is not well-understood. Recent data suggest the complex interaction of tropoelastin, the monomeric form of elastin, with a number of other elastic matrix-associated proteins, including fibrillins, fibulins, and matrix-associated glycoprotein (MAGP), is important to achieve the proper architecture of the elastic matrix. At the same time, it is becoming clear that self-assembly properties intrinsic to tropoelastin itself, reflected in a temperature-induced phase separation known as coacervation, are also important in this assembly process. In this study, using a well-characterized elastin-like polypeptide that mimics the self-assembly properties of full-length tropoelastin, the process of self-assembly is deconstructed into "coacervation" and "maturation" stages that can be distinguished kinetically by different parameters. Members of the fibrillin, fibulin, and MAGP families of proteins are shown to profoundly affect both the kinetics of self-assembly and the morphology of the maturing coacervate, restricting the growth of coacervate droplets and, in some cases, causing clustering of droplets into fibrillar structures.

Elastin is a major structural component of large elastic arteries and a principal determinant of arterial biomechanical properties. Elastin loss-of-function mutations in humans have been linked to the autosomal-dominant disease supravalvular aortic stenosis, which is characterized by stenotic lesions in both the systemic and pulmonary circulations. To better understand how elastin insufficiency influences the pulmonary circulation, we evaluated pulmonary cardiovascular physiology in a unique set of transgenic and knockout mice with graded vascular elastin dosage (range 45-120% of wild type). The central pulmonary arteries of elastin-insufficient mice had smaller internal diameters (P < 0.0001), thinner walls (P = 0.002), and increased opening angles (P = 0.002) compared with wild-type controls. Pulmonary circulatory pressures, measured by right ventricular catheterization, were significantly elevated in elastin-insufficient mice (P < 0.0001) and showed an inverse correlation with elastin level. Although elastin-insufficient animals exhibited mild to moderate right ventricular hypertrophy (P = 0.0001) and intrapulmonary vascular remodeling, the changes were less than expected, given the high right ventricular pressures, and were attenuated compared with those seen in hypoxia-induced models of pulmonary arterial hypertension. The absence of extensive pathological cardiac remodeling at the high pressures in these animals suggests a developmental adaptation designed to maintain right-sided cardiac output in a vascular system with altered elastin content.

Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to alpha-L-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via mechanisms that are unclear, but may result from the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Elastin fragmentation causes aortic dilatation and valvular insufficiency, which can result in cardiovascular disease. The pathophysiology of aortic disease was evaluated in MPS I mice. MPS I mice have normal elastic fiber structure and aortic compliance at early ages, which suggests that elastin assembly is normal. Elastin fragmentation and aortic dilatation are severe at 6 months, which is temporally associated with marked increases in mRNA and enzyme activity for two elastin-degrading proteins, matrix metalloproteinase-12 (MMP-12) and cathepsin S. Upregulation of these genes likely involves activation of STAT proteins, which may be induced by structural stress to smooth muscle cells from accumulation of glycosaminoglycans in lysosomes. Neonatal intravenous injection of a retroviral vector normalized MMP-12 and cathepsin S mRNA levels and prevented aortic disease. We conclude that aortic dilatation in MPS I mice is likely due to degradation of elastin by MMP-12 and/or cathepsin S. This aspect of disease might be ameliorated by inhibition of the signal transduction pathways that upregulate expression of elastase proteins, or by inhibition of elastase activity. This could result in a treatment for patients with MPS I, and might reduce aortic aneurism formation in other disorders.

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.

Diseases linked to the elastin gene arise from loss-of-function mutations leading to protein insufficiency (supravalvular aortic stenosis) or from missense mutations that alter the properties of the elastin protein (dominant cutis laxa). Modeling these diseases in mice is problematic because of structural differences between the human and mouse genes. To address this problem, we developed a humanized elastin mouse with elastin production being controlled by the human elastin gene in a bacterial artificial chromosome. The temporal and spatial expression pattern of the human transgene mirrors the endogenous murine gene, and the human gene accurately recapitulates the alternative-splicing pattern found in humans. Human elastin protein interacts with mouse elastin to form functional elastic fibers and when expressed in the elastin haploinsufficient background reverses the hypertension and cardiovascular changes associated with that phenotype. Elastin from the human transgene also rescues the perinatal lethality associated with the null phenotype. The results of this study confirm that reestablishing normal elastin levels is a logical objective for treating diseases of elastin insufficiency such as supravalvular aortic stenosis. This study also illustrates how differences in gene structure and alternative splicing present unique problems for modeling human diseases in mice.

Mice heterozygous for the elastin gene (ELN(+/-)) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN(+/-) mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN(+/-) cardiovascular system, we increased blood pressure in adult ELN(+/+) and ELN(+/-) mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN(+/+) and ELN(+/-)-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN(+/+) and ELN(+/-) hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN(+/-) mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.

Cigarette smoking is the strongest risk factor for emphysema. However, sensitivity to cigarette smoke-induced emphysema is highly variable, and numerous genetic and environmental factors are thought to mitigate lung response to injury. We report that the quantity of functional elastin in the lung is an important modifier of both lung development and response to injury. In mice with low levels of elastin, lung development is adversely affected, and mice manifest with congenital emphysema. Animals with intermediate elastin levels exhibit normal alveolar structure but develop worse emphysema than normal mice following cigarette smoke exposure. Mechanical testing demonstrates that lungs with low levels of elastin experience greater tissue strains for any given tissue stress compared with wild-type lungs, implying that force-mediated propagation of lung injury through alveolar wall failure may worsen the emphysema after an initial enzymatic insult. Our findings suggest that quantitative deficiencies in elastin predispose to smoke-induce emphysema in animal models and suggest that humans with altered levels of functional elastin could have relatively normal lung function while being more susceptible to smoke-induced lung injury.

Mice lacking macrophage elastase (matrix metalloproteinase-12, or MMP-12) were previously shown to be protected from the development of cigarette smoke-induced emphysema and from the accumulation of lung macrophages normally induced by chronic exposure to cigarette smoke. To determine the basis for macrophage accumulation in experimental emphysema, we now show that bronchoalveolar lavage fluid from WT smoke-exposed animals contained chemotactic activity for monocytes in vitro that was absent in lavage fluid from macrophage elastase-deficient mice. Fractionation of the bronchoalveolar lavage fluid demonstrated the presence of elastin fragments only in the fractions containing chemotactic activity. An mAb against elastin fragments eliminated both the in vitro chemotactic activity and cigarette smoke-induced monocyte recruitment to the lung in vivo. Porcine pancreatic elastase was used to recruit monocytes to the lung and to generate emphysema. Elastin fragment antagonism in this model abrogated both macrophage accumulation and airspace enlargement.

We describe a novel variant in the terminal exon of human elastin, c.2318 G > A, resulting in an amino acid substitution of glycine 773 to aspartate (G773D) in a pedigree with severe early-onset chronic obstructive pulmonary disease (COPD). Transfection studies with elastin cDNAs demonstrate that the glycine to aspartate change compromises the ability of the mutant protein to undergo normal elastin assembly. Other functional consequences of this amino acid substitution include altered proteolytic susceptibility of the C-terminal region of elastin and reduced interaction of the exon 36 sequence with matrix receptors on cells. These results suggest that the G773D variant confers structural and functional consequences relevant to the pathogenesis of COPD.

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months "recovery" in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months "recovery" in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

Supravalvular aortic stenosis (SVAS) is associated with decreased elastin and altered arterial mechanics. Mice with a single deletion in the elastin gene (ELN(+/-)) are models for SVAS. Previous studies have shown that elastin haploinsufficiency in these mice causes hypertension, decreased arterial compliance, and changes in arterial wall structure. Despite these differences, ELN(+/-) mice have a normal life span, suggesting that the arteries remodel and adapt to the decreased amount of elastin. To test this hypothesis, we performed in vitro mechanical tests on abdominal aorta, ascending aorta, and left common carotid artery from ELN(+/-) and wild-type (C57BL/6J) mice. We compared the circumferential and longitudinal stress-stretch relationships and residual strains. The circumferential stress-stretch relationship is similar between genotypes and changes <3% with longitudinal stretch at lengths within 10% of the in vivo value. At mean arterial pressure, the circumferential stress in the ascending aorta is higher in ELN(+/-) than in wild type. Although arterial pressures are higher, the increased number of elastic lamellae in ELN(+/-) arteries results in similar tension/lamellae compared with wild type. The longitudinal stress-stretch relationship is similar between genotypes for most arteries. Compared with wild type, the in vivo longitudinal stretch is lower in ELN(+/-) abdominal and carotid arteries and the circumferential residual strain is higher in ELN(+/-) ascending aorta. The increased circumferential residual strain brings the transmural strain distribution in ELN(+/-) ascending aorta close to wild-type values. The mechanical behavior of ELN(+/-) arteries is likely due to the reduced elastin content combined with adaptive remodeling during vascular development.

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury.

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln+/-) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln+/- animals were found to be stably hypertensive from birth, with a mean arterial pressure 25-30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln+/- arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln+/- mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the renin-angiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension.

This study evaluates possible changes in the synthesis/degradation of elastic components of the vein wall in an attempt to explain the development of varicosis. Healthy and varicose saphenous veins were subjected to immunohistochemical analysis using anti-elastin, anti-fibrillin-1, anti-elastase, anti-transforming growth factor (TGF)-beta and anti-latent TGFbeta binding protein (LTBP)-2 monoclonal antibodies. In situ hybridization was performed using specific probes for tropoelastin and fibrillin-1. In healthy veins, elastin and fibrillin-1 showed even, overlapping distribution patterns indicating their particular abundance in the adventitia and at the intima/media interface. The expression of tropoelastin and fibrillin-1 was high in smooth muscle cells bordering the elastic laminae. Elastin, fibrillin-1, and cells expressing fibrillin-1 and tropoelastin mRNA showed a patchy disorganized pattern, particularly in the proximal varicose segments of patients under 50 years of age. Enhanced elastase activity was noted in both control and varicose specimens from elderly subjects. Varicose veins specimens showed greater LTBP-2 and TGF expression. Both molecules were detected in the subendothelium and the media, particularly in areas of marked injury. Our findings suggest that the development of the varicose condition involves a restructuring of the elastic component of the vein wall, perhaps as a consequence of changes in the transcription mechanisms of muscle layer cells.

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.

1. Elastin was isolated from the bulbus arteriosus of a salmonid fish. Monoclonal and polyclonal antibodies, elicited against a CNBr digest of this protein, immunoprecipitated a polypeptide of Mr 43,000 from fish cell culture medium. 2. Cell-free translation of salmon poly A+ RNA produced a protein of approximately 43 kD that was immunoprecipitated with anti-elastin antibodies. The corresponding mRNA had an approximate Mr of 2 kb. 3. Despite similarities in amino acid composition, the differences in Mr between mammalian and salmon mRNA and protein suggest a divergence of fish and higher vertebrate elastins from an earlier ancestral gene.

Abdominal aortic aneurysms (AAA) are localized expansions of the abdominal aorta that develop due to chronic proteolytic disruption of the structural extracellular matrix (ECM) components (elastin and collagen) within the aorta wall. Major limitations in arresting or reversing AAAs lie in naturally poor and aberrant regeneration and repair of elastic matrix structures in the aorta wall. Bone marrow derived mesenchymal stem cells (BM-MSCs) have emerged as a promising regenerative tool and their therapeutic effects are also known to be effected through their paracrine secretions. Extracellular vesicles (EVs) present in these secretions have emerged as critical cellular component in facilitating many therapeutic benefits of MSCs. EV treatment is thus potentially appealing as a stem cell-inspired cell-free approach to avoid possible phenotypic plasticity of MSCs in vivo. In this study, we investigated the thus far unknown effects of BM-MSC derived EVs on vascular elastic matrix repair in the context of AAA treatment. EVs isolated from BM-MSC source were characterized and their pro-regenerative and their anti-proteolytic effects were evaluated on our established in vitro experimental conditions derived from AAA rat model. Our studies revealed the efficacy of BM-MSC derived EVs in attenuating the proteolytic activity and also in imparting elastic matrix regenerative benefits under aneurysmal environment. Interestingly, compared to cell culture conditioned media (CCM), EVs demonstrated superior regenerative and anti-proteolytic benefits in a proteolytic injury culture model of AAA. From these studies, it appears that EVs derived from BM-MSCs could be beneficial in undertaking a reparative effort in AAA induced degeneration of vascular tissue. Statement of Significance Abdominal aortic aneurysms (AAAs) are localized, rupture-prone expansions of the aorta which result from loss of wall flexibility due to enzymatic breakdown of elastic fibers. There are no established alternatives to surgery, which possess high risk for the mostly elderly patients. Our previous studies have established the elastic regenerative and reparative effect of cell culture secretions derived from adult stem cell source. In this study, we propose to isolate extracellular vesicles (exosomes) from these secretions and evaluate their regenerative benefits in AAA smooth muscle cell culture model. This simple and innovative treatment approach has the potential to arrest or reverse AAA growth to rupture, not possible so far. Image, graphical abstract [ABSTRACT FROM AUTHOR]